Combined effects of novel tyrosine kinase inhibitor AMN107 and histone deacetylase inhibitor LBH589 against unmutated or mutant Bcr-Abl-expressing human leukemia cells

6592 Background: AMN107 has potent in vitro and in vivo activity against the un-mutated and most mutant forms of Bcr-Abl tyrosine kinase (TK). We have previously demonstrated that in CML cells the pan-histone deacetylase inhibitor LBH589 depletes both unmutated and mutated Bcr-Abl, as well as attenuates p-AKT and Raf-1 levels. In the present studies we determined the effects of AMN107 and/or LBH589 in unmutated or mutated Bcr-Abl-expressing mouse BaF3 cells, or human CML K562 and LAMA-84 cells, as well as in primary CML cells procured from patients with imatinib mesylate (IM)-refractory CML. Methods: Cultured and primary CML cells were exposed to 20 to 100 nM AMN107 and/or LBH589 for 24 to 48 hours. Following this, the % of apoptotic cells was determined, as well as immunoblot analyses were performed to determine Bcr-Abl, p-STAT5, p-AKT, p-CrkL, Bim, Bcl-xL and p27 levels. Synergy between the apoptotic effects was determined by the median dose-effect isobologram analysis. Results: AMN107 was 20-fold more potent than IM, and was more potent in inhibiting Bcr-Abl TK activity and attenuating p-STAT5, p-AKT, Bcl-xL, and c-Myc levels in K562 and LAMA-84 cells. Co-treatment with LBH589 and AMN107 exerted synergistic apoptotic effects against human CML (but not the normal bone marrow progenitor cells), with more attenuation of p-STAT5, p-ERK1/2, c-Myc and Bcl-xL and increased p27 and Bim levels. LBH589 attenuated Bcr-Abl levels and induced apoptosis of mouse pro-B BaF3 cells containing ectopic expression of unmutated Bcr-Abl or the IM-resistant Bcr-AblT315I and Bcr-AblE255K. Treatment with LBH589 also depleted Bcr-Abl levels and induced apoptosis in IM-resistant primary human CML cells, including those with expression of Bcr-AblT315I. As compared to either agent alone, co-treatment with AMN107 and LBH589 induced more loss of cell viability of primary IM-resistant CML cells. Conclusions: These findings support in vivo testing of the combination of LBH589 and AMN107 against IM naïve or resistant CML, with the goal to eradicate IM sensitive or resistant CML. [Table: see text]

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Combined effects of novel tyrosine kinase inhibitor AMN107 and histone deacetylase inhibitor LBH589 against Bcr-Abl–expressing human leukemia cells

Blood ◽

10.1182/blood-2005-11-4639 ◽

2006 ◽

Vol 108 (2) ◽

pp. 645-652 ◽

Cited By ~ 108

Author(s):

Warren Fiskus ◽

Michael Pranpat ◽

Purva Bali ◽

Maria Balasis ◽

Sandhya Kumaraswamy ◽

...

Keyword(s):

Tyrosine Kinase ◽

Histone Deacetylase ◽

Histone Deacetylase Inhibitor ◽

Kinase Inhibitor ◽

Ectopic Expression ◽

Myelogenous Leukemia ◽

Human Leukemia ◽

Deacetylase Inhibitor ◽

Induced Apoptosis

AMN107 (Novartis Pharmaceuticals, Basel, Switzerland) has potent in vitro and in vivo activity against the unmutated and most common mutant forms of Bcr-Abl. Treatment with the histone deacetylase inhibitor LBH589 (Novartis) depletes Bcr-Abl levels. We determined the effects of AMN107 and/or LBH589 in Bcr-Abl–expressing human K562 and LAMA-84 cells, as well as in primary chronic myelogenous leukemia (CML) cells. AMN107 was more potent than imatinib mesylate (IM) in inhibiting Bcr-Abl tyrosine kinase (TK) activity and attenuating p-STAT5, p-AKT, Bcl-xL, and c-Myc levels in K562 and LAMA-84 cells. Cotreatment with LBH589 and AMN107 exerted synergistic apoptotic effects with more attenuation of p-STAT5, p-ERK1/2, c-Myc, and Bcl-xL and increases in p27 and Bim levels. LBH589 attenuated Bcr-Abl levels and induced apoptosis of mouse pro-B BaF3 cells containing ectopic expression of Bcr-Abl or the IM-resistant, point-mutant Bcr-AblT315I and Bcr-AblE255K. Treatment with LBH589 also depleted Bcr-Abl levels and induced apoptosis of IM-resistant primary human CML cells, including those with expression of Bcr-AblT315I. As compared with either agent alone, cotreatment with AMN107 and LBH589 induced more loss of cell viability of primary IM-resistant CML cells. Thus, cotreatment with LBH589 and AMN107 is active against cultured or primary IM-resistant CML cells, including those with expression of Bcr-AblT315I.

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Pre-Clinical Efficacy of Combined Therapy with Novel β-Catenin Antagonist BC2059 and Histone Deacetylase Inhibitor Against Cultured and Primary AML Blast Progenitor Cells

Blood ◽

10.1182/blood.v120.21.287.287 ◽

2012 ◽

Vol 120 (21) ◽

pp. 287-287

Author(s):

Kapil Bhalla ◽

Warren Fiskus ◽

Stephen K. Horrigan ◽

Sunil Abhyankar ◽

Joseph McGuirk ◽

...

Keyword(s):

Cyclin D1 ◽

Progenitor Cells ◽

Histone Deacetylase ◽

Histone Deacetylase Inhibitor ◽

Nuclear Accumulation ◽

Hematopoietic Stem ◽

Tail Vein ◽

Deacetylase Inhibitor ◽

Induced Apoptosis

Abstract Abstract 287 The canonical WNT-β-catenin pathway is essential for self-renewal, growth and survival of AML stem and early blast progenitor cells (BPCs). Deregulated WNT signaling in AML inhibits polyubiquitylation and proteasomal degradation of β-catenin by the multi-protein complex. The inactivation of the degradation complex for β-catenin results in the preservation, nuclear translocation and activity of β-catenin in AML BPCs. This is especially notable in AML BPCs expressing FLT3-ITD, where increased activity of PI3K/AKT phosphorylates and inactivates GSK3β, thereby inhibiting phospho-degradation, increasing stability and nuclear accumulation of β-catenin. Activated FLT3 kinase has also been shown to directly phosphorylate β-catenin and promote its stabilization and nuclear localization. As a co-activator, in the nucleus β-catenin interacts with the bipartite T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factor, which increases the expression of pro-growth and pro-survival genes, including cyclin D1, Myc and survivin. Aberrant expression of LEF1 in hematopoietic stem cells has been shown to induce AML. β-catenin is also required for the HOXA9 and MEIS1 or MLL-AF9-mediated transformation of hematopoietic stem/progenitor cells. Thus, reactivation of β-catenin is required for maintenance of leukemic transformation making it an attractive drug target in AML. Here, we determined the in vitro and in vivo anti-AML activity of BC2059 (β-Cat Pharmaceuticals), a potent, small molecule, anthraquinone oxime-analog, against cultured (HL-60, OCI-AML3 and MV4-11 cells) and primary AML BPCs with or without the expression of FT3-ITD. First, as compared to the normal human CD34+ cells, FLT3-ITD expressing AML BPCs showed increased levels of β-catenin, mostly in the nucleus, as determined by confocal immunofluorescence microscopy. Exposure to 100 nM of BC2059 (BC) induced β-catenin degradation through the proteasome, as well as attenuated the nuclear and cytoplasmic levels of β-catenin in the cultured and primary AML BPC. Chromatin immunoprecipitation with anti-β-catenin antibody demonstrated that treatment with BC2059 (BC) reduced the binding of β-catenin to the WNT response elements (WRE) in the promoter DNA of its target genes, including Myc, cyclin D1 and survivin. Estimation of the intracellular luciferase levels in AML cells transfected with the TOP/FLASH versus FOP/FLASH construct showed that treatment with BC2059 significantly reduced only the TOP-FLASH luciferase activity, indicating that BC inhibits the expression of genes with promoters containing WRE elements. This was associated with reduced mRNA and protein levels of cyclin D1, MYC and survivin. Treatment with BC dose-dependently induced apoptosis of cultured and primary AML BPCs (up to 70%), including apoptosis of the CD34+CD38-Lin- AML BPCs. In contrast, BC induced apoptosis in < 10% of normal CD34+ progenitor cells. We have previously reported that treatment with the histone deacetylase inhibitor panobinostat (PS) attenuates p-FLT3, p-AKT and p-GSK3β levels in AML BPCs expressing FLT3-ITD. Consistent with this, here, we determined that co-treatment with BC and PS (10 to 20 nM) synergistically induced apoptosis of cultured and primary AML BPCs. Against primary AML BPCs expressing FLT3-ITD, co-treatment with the FLT3 kinase inhibitor AC220 (100 nM) further augmented BC mediated depletion of the cytoplasmic and nuclear levels of β-catenin and significantly enhanced BC-induced apoptosis (p < 0.01). This was associated with induction of BIM and p27 with depletion of MCL-1 levels. Following tail vein infusion and establishment of AML by OCI-AML3 cells in NOD-SCID mice, treatment with BC (5 or 10 mg/kg b.i.w, IV) for three weeks demonstrated improved survival of the mice compared to the vehicle control treated mice (p <0. 001). Survival was further improved upon co-treatment with BC and PS (5 mg/kg IP, MWF). BC treatment (5 or 10 mg/kg IV) also dramatically improved survival of the NOD/SCID/IL2Rγ-depleted mice with established human AML following tail-vein injection of primary AML BPCs expressing FLT3 ITD. Mice did not experience any toxicity or weight loss. These findings support a compelling rationale for further development and in vivo testing of the BC-based combination with PS and FLT3 antagonist against human AML BPCs. Disclosures: Horrigan: BetaCat Pharmaceuticals: Employment. Sharma:BetaCat Pharmaceuticals: Equity Ownership.

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Synergistic Cytotoxic Effects of a Combination of a Novel Tyrosine Kinase Inhibitor AMN107 and Histone Deacetylase Inhibitor LBH589 Against Bcr-Abl Expressing Human Leukemia Cells.

Blood ◽

10.1182/blood.v104.11.1977.1977 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1977-1977 ◽

Cited By ~ 3

Author(s):

Anna Scuto ◽

Srinivas Annavarapu ◽

Purva Bali ◽

Molly McCullers ◽

Warren Fiskus ◽

...

Keyword(s):

Cell Cycle ◽

Tyrosine Kinase ◽

Histone Deacetylase ◽

Histone Deacetylase Inhibitor ◽

G1 Phase ◽

Dependent Manner ◽

Wild Type ◽

Deacetylase Inhibitor ◽

Apoptotic Effects ◽

Phase Accumulation

Abstract AMN107 (Novartis Pharmaceuticals, Basel, Switzerland) is a potent aminopyrimidine tyrosine kinase (TK) inhibitor, which is active at nanomolar concentrations against c-Kit, PDGFR as well as the wild type Bcr-Abl and common variety of mutant Bcr-Abl (e.g., M351T, F317L and E255V) TK. In a previous report, we demonstrated that treatment with the hydroxamic acid analogue histone deacetylase inhibitor LBH589 (Novartis) (20 to 100 nM) induces hsp90 acetylation and promotes polyubiquitylation and proteasomal degradation of Bcr-Abl, which is associated with apoptosis of the cultured Bcr-Abl expressing human chronic myeloid leukemia-blast crisis (CML-BC) K562 and LAMA-84 cells, as well as of primary CML-BC cells. In the present studies, we determined the cell cycle and apoptotic effects of AMN107 and/or LBH589 in K562, LAMA-84 and primary CML-BC cells. Treatment with AMN107 (20 to 100 nM) induced cell cycle G1 phase accumulation and apoptosis, exerting 10 to 20 fold more potent effect than imatinib mesylate (IM) in K562 and LAMA-84 cells. This was associated with marked induction of p27, inhibition of Bcr-Abl TK activity, as well as attenuation of the levels of p-STAT5, p-AKT, Bcl-xL, and c-Myc levels in a dose dependent manner. Co-treatment with LBH589 (50 nM) and AMN107 (100 nM) induced more G1 phase accumulation than either agent alone. The combination of AMN107 and LBH589 also exerted synergistic apoptotic effects in K562 cells, as determined by the median effect isobologram analysis. This was associated with more attenuation of the levels of pro-growth and pro-survival proteins, e.g., p-STAT5, p-ERK1/2, c-Myc and Bcl-xL, as well as greater induction of the levels of pro-death p27 and Bim proteins. Treatment of human AML HL-60 cells containing ectopic expression of the IM-refractory, mutant Bcr-AblT315I with LBH589 (50 nM for 24 hours) attenuated the mutant Bcr-Abl levels and induced apoptosis of HL-60/Bcr-AblT315I cells. Treatment with AMN107 (up to 2.0 μM) alone was ineffective in inducing loss of viability of HL-60/Bcr-AblT315I cells, and co-treatment with LBH589 (50 nM) and AMN107 did not induce more loss of cell viability of HL-60/Bcr-AblT315I cells than treatment with LBH589 alone. AMN107 levels ≥ 200 nM significantly inhibited survival of HL-60/Bcr-AblE255K and HL-60/Bcr-AblM351T cells. Consistent with this, the combined treatment with LBH589 and AMN107 also induced more loss of viability than treatment with either agent alone in 4 samples of IM-refractory primary CML-BC cells. These studies demonstrate that both AMN107 and LBH589 are active against IM-resistant CML-BC and the combination of the two exerts superior activity against IM-sensitive and IM-resistant, wild type and mutant Bcr-Abl expressing cultured and primary CML cells.

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